It is essential for survival that a wound stops bleeding, i.e. that the body possesses an adequate mechanism for haemostasis. The process of blood clotting can be activated in the case of injuries or inflammations by either extrinsic or intrinsic factors, e.g. tissue factor (TF) or Hagemann factor (F XII), respectively. Both activation channels are continued in a common branch of the cascade resulting in thrombin formation. The thrombin itself finally initiates the formation of fibrin fibres which represent the protein backbone of blood clots.
The other main constituent of the final blood clot are the thrombocytes which are interconnected by the fibrin fibres and undergo a number of physiological changes during the process of coagulation. Within limits a lack of thrombocytes can be substituted by an increased amount of fibrin or vice versa. This is reflected in the observation that the thrombocyte counts as well as the fibrinogen concentration varies even within healthy patients.
Various methods have been introduced to assess the potential of blood to form an adequate clot and to determine the blood clots stability. Common laboratory tests such as thrombocyte counts or the determination of fibrin concentration provide information on whether the tested component is available in sufficient amount but lack in answering the question whether the tested component works properly under physiological conditions (e.g. the activity of fibrinogen under physiological conditions can not be accessed by common spectroscopic methods). Other common tests such as the prothrombin time (Quicktest) or the partial thromboplastin time (PTT) work on blood-plasma exclusively and therefore require an additional step for preparation and additional time which is unfavourable especially under POC (point of care) conditions.
Another group of tests which overcomes these problems is summarized by the term “viscoelastic methods”. The common feature of these methods is that the blood clot firmness (or other parameters dependent there on) is continuously determined, from the formation of the first fibrin fibres until the dissolution of the blood clot by fibrinolysis. Blood clot firmness is a functional parameter, which is important for haemostasis in vivo, as a clot must resist blood pressure and shear stress at the site of vascular injury. Clot firmness results from multiple interlinked processes: coagulation activation, thrombin formation, fibrin formation and polymerization, platelet activation and fibrin-platelet interaction and can be compromised by fibrinolysis. Thus, by the use of viscoelastic monitoring all these mechanisms of the coagulation system can be assessed.
A common feature of all these methods used for coagulation diagnosis is that the blood clot is placed in the space between a cylindrical pin and an axially symmetric cup and the ability of the blood clot to couple those two bodies is determined.
The first viscoelastic method was called “thrombelastography” (Hartert H: Blutgerinnungsstudien mit der Thrombelastographie, einem neuen Untersuchungsverfahren. Klin Wochenschrift 26:577-583, 1948). In the thromboelastography, the sample is placed in a cup that is periodically rotated to the left and to the right by about 5°, respectively. A pin is freely suspended by a torsion wire. When a clot is formed it starts to transfer the movement of the cup to the pin against the reverse momentum of the torsion wire. The movement of the pin as a measure for the clot firmness is continuously recorded and plotted against time. For historical reasons the firmness is given in millimeters.
The outcome of a typical measurement of this kind is illustrated in FIG. 1. One of the most important parameters is the time between the chemically induced start of the coagulation cascade and the time until the first long fibrin fibres have been build up which is indicated by the firmness signal exceeding a defined value. This parameter will be called clotting time or just CT in the following. Another important parameter is the clot formation time (CFT) which gives a measure for the velocity of the development of a clot. The CFT is defined as the time it takes for the clot firmness to increase from 4 to 20 mm. The maximum firmness a clot reaches during a measurement, further on referred to as maximum clot firmness or just MCF, is also of great diagnostic importance.
Modifications of the original thromboelastography technique (nowadays also called thromboelastometry) have been described by Cavallari et al. (U.S. Pat. No. 4,193,293), by Do et al. (U.S. Pat. No. 4,148,216), by Cohen (U.S. Pat. No. 6,537,819), by Hartert et al. (U.S. Pat. No. 3,714,815) and by Calatzis et al. (U.S. Pat. No. 5,777,215).
During coagulation the fibrin backbone creates a mechanical elastic linkage between the surfaces of the blood-containing cup and a pin plunged therein. A proceeding coagulation process induced by adding one or more activating factor(s) can thus be observed. In this way, various deficiencies of a patient's haemostatic status can be revealed and used for proper medical intervention.
A general advantage of thromboelastometry compared to other laboratory methods in this field therefore is that the coagulation process and the change of mechanical properties of the sample are monitored as a whole. This means that contrary to the other laboratory methods mentioned above, thromboelastometry does not only indicate if all components of the coagulation pathways are available in sufficient amounts but also if each component works properly.
To get detailed information on the correct amount and function of the thrombocytes as well as the fibrinogen and certain factors nowadays there is an increasing amount of chemicals available which activate or inhibit certain components of the coagulation system. This allows determining exactly at which point of the coagulation system a problem is located.
For practical reasons these chemicals are usually injected to the disposable plastic cup which later on is used for the measurement by using a pipette (either a manual or an automatic one). In the last preparation step, after the blood of plasma sample has been added, the whole amount of sample (blood/plasma and the additional chemicals) is mixed by drawing it into the pipette tip and dispensing it into the cup again.
The possibility to chemically activate or to disable certain components of the coagulation system is especially useful in conjunction with state-of-the-art thromboelastometers such as the ROTEM (Pentapharm GmbH, Munich, Germany) which allows conducting four measurements in parallel. This allows to achieve detailed information on the current status of the coagulation-situation of a patient and therefore allows an appropriate therapy within several minutes. Furthermore, the efficiency of a certain medication might be tested in vitro prior to the application to the patient.
This is of particular importance in case of patients struck by massive blood loss as it often occurs in context with multiple traumata. The blood of such patients often is diluted due to infusions which are administered to replace the loss in volume. This leads to a decrease of the concentration of thrombocytes as well as coagulation factors such as fibrinogen.
A topic of outstanding importance in this context is the determination of the fibrin networks contribution to the final stability of a growing blood clot. This can be achieved by adding a thrombocyte inhibitor, e.g. Cytochalisch D, to the sample before measurement. That way the activity of fibrin becomes directly accessible.
One problem in thromboelastometric measurements may result from decreasing signal to noise ratio if the total firmness of the sample becomes comparably low. This situation especially occurs for measurements in which the thrombocytes are chemically inactivated (such as mentioned above) because these tests naturally exhibit a low final firmness. The situation becomes even worse if the original blood sample is highly diluted due to the earlier addition of substitutes. Since a sufficient signal to noise ratio might be crucial for an appropriate fibrinogen medication (in particular to choose an appropriate amount of fibrinogen substitute) it would be an important achievement to increase the sensitivity of those tests.
The reason for the rather low signal to noise ratio when testing the fibrinogen function of pathologic samples originates from applying the thromboelastometric method near the lower limit of sensitivity: The geometry of the standardized disposables (the outer diameter of the pin is about 5.0 mm and the space between cup and pin is about 1.0 mm) and the amount of blood used per test were originally chosen to obtain best signals when measuring conventionally activated ‘full’ clots of non-pathologic blood samples. Such tests result in values for the maximum clot firmness (MCF) between 50 and 70 mm, which is the most sensitively detected range of the method. In thrombocyte inhibited tests, however, only the fibrinogen contribution to the clot is measured, since the platelet functionality is completely suppressed. Hence, these tests yield only MCF's between 15 and 25 mm for normal patients, while MCF's well below 10 mm are typically observed in the case of pathologic samples—with no definable lower limit. Considering a general sensitivity level of about 2 mm for the current disposable geometry, higher test-to-test result variations (coefficient of variations) are a consequence when measuring such samples.
The magnitude of the measured signal is proportional to the torque being transmitted to the shaft of the instrument by elastic fibrin fibres between cup and pin walls. Therefore it depends on the thickness of the blood clot on the total area of the clot surface.
As a conventional solution, the measurement signal could be increased by increasing the sample amount if the expected clot firmness is rather low. However, this approach limited to the amount of blood usually available for coagulation analysis in clinical practice. A further practical limitation of this approach is that the maximum increase of sample volume and cup is limited due to the geometric dimensions of the commercially available thromboelastometers. Furthermore, there are situations where the amount of blood available for analysis is further limited, especially in surgery on infants (due to ethical considerations) or in pharmaceutical industry where mice are used as donors. Beyond that, thromboelastography in the pharmaceutical industry for drug development has an increased demand for extremely high accuracy measurements on small samples.
To reach maximum accuracy, it is desirable to achieve that the transfer of torque to the instrument shaft is shifted to the most sensitive range of the instrument for each test.
The purpose of this invention is to achieve this by optimization of the geometry of cups and/or pins intended for tests with currently low signal amplitudes. Another field of application would be the pharmacological industry or any situation where the available amount of sample is limited.
It is therefore an object of the present invention to provide an apparatus for measuring the coagulation characteristics of a test liquid, whereby the ratio between signal amplitude and the needed amount of test liquid is increased. It is also an object of the invention to provide a method for measuring the coagulation characteristics of a test liquid by means of such an apparatus.
The object is attained by an apparatus comprising the features disclose herein, in particular an apparatus comprising measuring coagulation characteristics of a test liquid, in particular of a blood sample, comprising: a cup for receiving said test liquid; a pin having a head portion suitable to be immersed into said test liquid of said cup; wherein said cup comprises at least one test liquid duct portion; wherein said head portion of said pin comprises at least one test liquid contacting portion, wherein each test liquid contacting portion is associated to and placeable inside the respective test liquid duct portion of said cup such that the lateral surfaces of the respective test liquid contacting portion of said pin and side walls of the associated test liquid duct portion of said cup are forming a test liquid gap there between having a predetermined width; and wherein said at least one test liquid duct portion and said associated test liquid contacting portion are shaped as a ring-segment, a method for measuring the coagulation characteristics of a test illiquid in particular a blood sample, via such an apparatus comprising: (a) measuring oscillation movement signal values by using said cup and said pin having predetermined geometrical dimensions; and (b) determining the coagulation characteristics of the test liquid using said signal values and a data carrier comprising a non-transient computer-readable medium containing code for executing this method.
Depending on the practical situation the present invention can be used either to increase the signal/noise ratio especially of those coagulation tests which provide only small signals in conventional thromboelastometry (such as test where the blood sample is treated with substances which deactivate the thrombocytes to enable the measurement of the fibrin contribution to the clot firmness solely as mentioned above), or just to decrease the amount of blood which is needed for the test.
The last point is of paramount importance in pharmacology because it would allow repeatable experiments with the blood of a single small laboratory animal such as a mouse. So far there are only two ways to overcome the problem of collecting enough mouse blood for thromboelastography: Pooling of small blood samples (50-100 μl) of several individuals or taking nearly the entire amount of blood of a single individual. The first approach has the disadvantage of averaging out the individual response of each mouse and therefore provides a result that only represents the average values of a multitude of samples. Under these conditions those results which occur only rarely (e.g. one pathological case in a sample consisting of the blood of 10 or more individuals) might not be detectable. The second approach of taking 300 μl would surely be lethal to small animals like mice or the same. This makes it impossible to compare samples taken from the same individual at different points of time, e.g. to monitor the success of a certain treatment or medication with time. Considering the partially high costs for special breeding of laboratory animals also financial considerations make it desirable reduce the amount of animals needed. Furthermore, less animals loose their lives in view of ethical aspects.